SOUTHERN BLOT

PCR is used to amplify DNA, restriction enzyme is present and electrophoresis is done

Gel placed in alkali solution which breaks hydrogen bonds causing the two strands to seperate

nylon sheet is placed on top of gell, alkali solution is drawn up through gel to paper towel by capillary action, bringing the DNA with it. negatively-chraged DNA sticks to positively-charges nylon membrane

nylon treated with UV light to fix DNA molecules

placed in a bag, probes anneal to DNA. which forms a hybrid DNA molecule

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GENETIC FINGERPRINTING

Difference found in non-coding DNA

non-coding DNA is mainly made up of long repetitive sequence

Amplified DNA of VNTR region is digested by restriction enzymes. Same recognition sequence should be present in different sample but lenght will depend on how many repeats

Run down on an electrophoresis gel to seperate fragment

visualised by southern blot

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VECTORS

In genetic engineering a vector is a lenght of DNA that carries the gene we want into a host cell

Plasmid are commonly used vectors

They are small so easy yo handle in test tube

Restriction enzymes alcut the gene from donor DNA

Same restriction enzyme used to cut in middle of one marker gene

Will anneal due to complementary sticky ends

DNA ligase will also be used

Hybrid DNA is produced

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MAKER GENES

Maker genes are used to find which cells have actually taken up the hybrid vector.

First maker genes distinguishes between cells that have taken up the plasmid from those who havent

Second will distinguish between cell that have taken up the hybrid and original plasmid.

test is done on replica plate

Colonies that grow on the first (tetracyljne) plate but not on the replica (ampicillin) plate are the ones we want as it has hybrid DNA.